Relationships between oxidative stress markers and red blood cell characteristics in renal azotemic dogs

Abstract

Oxidative stress parameters and erythrocyte characteristics were studied in 15 normal healthy dogs and 33 renal azotaemic dogs from Small Animal Hospital, Faculty of Veterinary Science, Chulalongkorn University. Dogs with renal azotaemia had reduced mean corpuscular volume (MCV) (P < 0.01), packed cell volume (PCV) (P < 0.001) and increased mean corpuscular hemoglobin concentration (MCHC) (P < 0.001). The relationship was found between degree of azotaemia and MCV, PCV and MCHC. Dogs with severe renal azotaemia had higher intraerythrocytic sodium contents (RBC-Na) (P < 0.05). The red blood cell catalase activity and glutathione and plasma malondialdehyde were unaltered while urinary malondialdehyde-creatinine ratio (U-MDA/Cr) increased significantly (P < 0.001). The U-MDA/Cr was correlated significantly with plasma creatinine concentration (P < 0.05), urinary protein-creatinine ratio (P < 0.05) and fractional excretion of sodium (P < 0.001). The results suggest some changes in RBC characteristics and urine oxidative stress marker in renal azotaemic dogs. Moreover, the U-MDA/Cr is a sensitive biochemical parameter which increased along with degree of renal dysfunction.

Introduction

Reduced glutathione (GSH), catalase activity (CAT) and MDA (expressed as a thiobarbituric acid reactive substances; TBARS) are important markers which were widely used to demonstrate oxidative stress condition. GSH, found mainly inside the cell, is antioxidant agent while catalase is a cytoplasmic enzyme responsible for hydrogen peroxide detoxification. Likewise, MDA is a product of polyunsaturated fatty acid degradation which releases into plasma as a result of membrane damage. Measurement of changes in these parameters may reflect tissue damage by oxidative stress. Increased reactive oxygen species (ROS) during cellular injury occurs in many pathological conditions in human such as atherosclerosis (Vigna et al., 2004), acute post-streptococcal glomerulonephritis (Devasena et al., 2001), malignant lymphoma (Abou-seif et al., 2000), cervical cancer (Kolanjiappan et al., 2002) and in renal ischemia/reperfusion injury (Sehirli et al., 2003). There are many clinical renal disease entities in which oxidants are considered to be pathogenic (Ichikawa et al., 1994). During tissue hypoxia and reperfusion, free radical-mediated cellular damage can be expected to occur and the formation exceeds the cellular detoxification capacity. Increased free radical formation enhances the activity of phospholipase which subsequently destroys lipid plasma membrane. Impairs of cellular volume and monovalent cation homeostasis between plasma membrane were suggested. Therefore, changes in intraerythrocytic sodium (Na) and potassium (K) and mean corpuscular volume and osmotic fragility were suggested during oxidative stress. Increased RBC-Na with decreased RBC-K was found in patient with CRF (Prasad et al., 1996) which may be related to suppression of Na/K pump at RBC membrane (Fervenza et al., 1989).

Anemia is usually found in dogs with chronic renal impairment which is due to lacking of hormone erythropoietin (EPO). However, anemic condition may not solely be due to suppression of red blood cell production but may be due to suppression of erythropoiesis by uraemic toxin or increased hemolysis (Sexuer and Matson, 1981). The mean osmotic fragility was greater in human with chronic hemodialyzed patients before dialysis than control healthy volunteer (Wu et al., 1998) but unchanged in renal failure dogs (King et al., 1992). Moreover, previous studies showed relationship between osmotic fragility and oxidative stress. Increased hydrogen peroxide-induced hemolysis with reduced RBC–GSH concentration was demonstrated in rats exposed to 80% oxygen (Webster et al., 1987). Elevated RBC-CAT with increased osmotic fragility in human with malignant lymphoma was also observed (Abou-Seif et al., 2000). From these data, changes in RBC characteristics in CRF may be related to oxidative stress. Increased plasma MDA with a reduction in RBC–GSH was found in human with CRF (Ozden et al., 2002). In dogs with kidney diseases, increased plasma MDA with decreased RBC superoxide dismutase, glutathione peroxidase and CAT activities were observed (Kargin and Fidanci, 2001). Recently, urinary MDA was suggested to be superior as a biochemical marker for renal lipid peroxidation. One study showed an increase in urinary MDA in burned patient with renal dysfunction (Kang et al., 2001).

Therefore, the objectives of this study were, first, to determine whether oxidative stress markers (RBC–GSH, RBC-CAT, plasma MDA and urinary MDA creatinine ratio (U-MDA/Cr) and RBC characteristics (RBC-Na, RBC-K, mean osmotic fragility, mean corpuscular volume and packed cell volume) were changes in dogs with renal azotaemia compared with control healthy dogs. Second, to determine the relationship among the oxidative stress markers, RBC characteristics with degree of renal impairment.